Substances addressing the opioid system are widely used pharmaceuticals; agonists (narcotic analgesics) for the treatment of chronic pain; antagonists generally for the treatment of addictive disorders, including substance abuse (opiate, alcohol, amphetamine) and non-substance, i.e. behavioral addictions. Given the difficulties associated with opioid agonist therapy, high risk of death due to overdose (more victims were reported in 2008, than overdose of heroin and cocaine combined) and the development of tolerance and addiction, there is a need for safer narcotic analgesics. The proposed research program uses structural, computational, biophysical, biochemical, and pharmacological approaches: (1) to develop a new level of understanding of the action the opioid receptors, | j , 6, K, and the nociceptin receptor, defining the structural basis for molecuar recognition and activation, and (2) to design new binding ligands. Aims are to (1) understand opioid receptor molecular recognition and functional selectivity using X-ray crystallography and NMR; (2) understand the molecular mechanisms for functional and pharmacological selectivity for the four receptors using computational approaches; and (3) understand ligand-directed signaling (G-protein-, arrestin-, JNK-dependent) and its relevance in mediating opioid receptor action. Three projects supported by four core groups via inter-project collaborations will address important but difficult questions at a scale not imagined prior to this year. Project 1 will carry ut structural determination on ligand bound complexes. Project 2 will carry out computational studies to define the binding pockets of opioid receptors and perform computer-assisted SBDD to design a new series of optimized compounds including allosteric, functionally selective and bitopic ligands. Project 3, will carry out in vitro and in vivo studies to characterize pharmacological properties of ligands and generate new receptor biased mutants to test hypotheses related to functional selectivity and opioid receptor actions. Core groups support the aims of all 3 projects and will be responsible for (1) large-scale production of purified opioid receptors; (2) synthesis of opiate ligands including newly designed tool compounds for structural and functional studies; (3) molecular profiling and screening of new small molecules, and (4) management and administration of the overall program. Facility resources will be provided by the NIGMS GPCR Networks' Structural Determination Pipeline for large-scale protein production and structure solution and the NIMH Psychoactive Drug Screening Program resources for selectivity profiling of novel small molecules.